Structural insights into the γ-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans

Abstract

(+)-γ-lactamase catalyzes the specific hydrolysis of (+)-γ-lactam out of the racemic γ-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (−)-γ-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the γ-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-γ-lactamase activity, which constitutes a novel family of γ-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (−)-γ-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-γ-lactam bound, and (−)-γ-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (−)-γ-lactam resemble those of IHLs, but the “cover” loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (−)-γ-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of γ-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

“…Structural analysis indicates that MhIHL lacks a loop in the entrance to the binding pocket compared to other IHLs. We suggest that this open conformation of the active cavity in MhIHL would facilitate both the binding of the substrate and the release of the product molecule but simultaneously reduce the specificity of the substrate, consistent with the comparable enzymatic kinetic parameters of the two enantiomers catalyzed by MhIHL (31). This promiscuous enzyme displays 10 times higher (ϩ)-␥-lactamase activity than the most active (ϩ)-␥-lactamase reported before (Table 1), making it a very good target for the preparation of enantiopure (Ϫ)-Vince lactam.…”

“…MhIHL has the conserved catalytic triad D13-K78-C111, with the C111 acting as the nucleophile. The proposed mechanism resembles the mechanism proposed for other ␣/␤-hydrolase enzymes, such as those from Acinetobacter baumannii PncA26 (31). Structural analysis indicates that MhIHL lacks a loop in the entrance to the binding pocket compared to other IHLs.…”

“…A ␥-lactamase from Microbacterium hydrocarbonoxydans (designated MhIHL) with high enzyme activity is the subject of the present study (16,31). MhIHL is a small enzyme with a molecular mass of 20 kDa.…”

“…It is assigned to the isochorismatase-like hydrolase (IHL) superfamily/cysteine hydrolase family, CDD classification cd00431 (32). Like other IHLs, MhIHL folds into a typical ␣/␤-fold with a six-stranded parallel ␤-sheet in the middle, flanked by three helices, and a single long helix on both sides of the sheet (31). MhIHL has the conserved catalytic triad D13-K78-C111, with the C111 acting as the nucleophile.…”

“…This promiscuous enzyme displays 10 times higher (ϩ)-␥-lactamase activity than the most active (ϩ)-␥-lactamase reported before (Table 1), making it a very good target for the preparation of enantiopure (Ϫ)-Vince lactam. A previous study showed that MhIHL could catalyze the hydrolysis of both enantiomers with a specific hydrolysis curve (31); thus, it is necessary to improve its enantioselectivity before it can be considered for practical applications. Another property which needs to be tailored is its poor thermostability.…”